Structural and Metal Ion Effects on Human Topoisomerase IIÎ±

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Structural and Metal Ion Effects on Human Topoisomerase II# Inhibition by #-(N)-Heterocyclic Thiosemicarbazones William H. Morris, Lana Ngo, James T Wilson, Wathsala Medawala, Anthony Brown, Jennifer D Conner, Florence Fabunmi, Derek J Cashman, Edward C Lisic, Tao Yu, Joseph E. Deweese, and Xiaohua Jiang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00204 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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Chemical Research in Toxicology

Structural and Metal Ion Effects on Human Topoisomerase IIα Inhibition by α-(N)-Heterocyclic Thiosemicarbazones

William H. Morris,§ Lana Ngo,§ James T. Wilson,‡ Wathsala Medawala,# Anthony Brown,§ Jennifer D. Conner,§ Florence Fabunmi,§ Derek J. Cashman, § Edward C. Lisic,§ Tao Yu,§ Joseph E. Deweese,‡¶* Xiaohua Jiang§*

§Department

of Chemistry, Tennessee Technological University, Cookeville, Tennessee 38505, United States

#Department

of Chemistry, Georgia College, Milledgeville, Georgia 31061, United States

‡Department

of Pharmaceutical Sciences, Lipscomb University College of Pharmacy and Health Sciences, Nashville, Tennessee 37204-3951, United States ¶Department

of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States

E-mail: [email protected]. Phone: 931-372-3814. Fax: 931-372-3434. Or [email protected], Phone: 615-966-7101. Fax: 615-966-7163.

Running title: The Pd(II) and Cu(II) thiosemicarbazone complexes inhibit DNA relaxation by topoisomerase IIα

ACS Paragon Plus Environment

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ABSTRACT Our previous research has shown that α-(N)-heterocyclic thiosemicarbazone (TSC) metal complexes inhibit human topoisomerase IIα (TopoIIα), while the ligands without metals do not. To find out the structural elements of TSC that are important for inhibiting TopoIIα, we have synthesized two series of α-(N)heterocyclic TSCs with various substrate ring segments, side chain substitutions and metal ions, and we have examined their activities in TopoIIα-mediated plasmid DNA relaxation and cleavage assays. Our goal is to explore the structure-activity relationship of α-(N)-heterocyclic TSCs and their effect on TopoIIα. Our data suggest that, similar to Cu(II)-TSCs, Pd(II)-TSC complexes inhibit plasmid DNA relaxation mediated by TopoIIα. In TopoIIα-mediated plasmid DNA cleavage assay, the Cu(II)-TSC complexes induce higher levels of DNA cleavage than their Pd(II) counterparts. The Cu(II)-TSC complexes with methyl, ethyl and tert-butyl substitutions are slightly more effective than those with benzyl and phenyl groups. The α-(N)-heterocyclic ring substrates of the TSCs, including benzoylpyridine, acetylpyridine, and acetylthiazole, do not exhibit a significant difference in TopoIIα-mediated DNA cleavage. Our data suggests that the metal ion of TSC complexes plays a predominant role in inhibition of TopoIIα, the side chain substitution of the terminal nitrogen plays a secondary role, while the substrate ring segment has the least effect. Our molecular modeling data support the biochemical data, which together provide a



mechanism by which Cu(II)-TSC complexes stabilize TopoIIα-mediated cleavage complexes.

Introduction Thiosemicarbazones (TSCs) are a group of compounds with an N-N-S tridentate coordination scaffold, which are excellent metal chelating agents. They were found to have antileukemic activities and antiproliferative activities against many cancer cell lines and transplanted murine tumors, making them promising candidates for chemotherapeutics.1,

2

A special group, the α-(N)-heterocyclic

TSCs, exhibits significant anticancer activities.3-5 One of the compounds from this group, 3-aminopyridine-2-carboxaldehyde TSC (Triapine) has been tested extensively in phase I/II clinical trials.6-9 The molecular mechanisms of how TSCs suppress tumor cell growth are quite complicated. Recent studies showed that an α-(N)-heterocyclic TSC compound, Dp44mT, demonstrates selective activities against cancer cells attributed to inhibition of topoisomerase IIα (TopoIIα).10 Several other α-(N)heterocyclic TSCs and Cu(II)-TSC complexes also exhibit similar inhibition against TopoIIα.11-15 For example, quinoline-2-carboxaldehyde TSCs and their Cu(II) and Ni(II) complexes have been confirmed as TopoIIα inhibitors.11, 13 Other metal-TSC complexes including Pd(II), Pt(II), Ga(III) and Ru(II) were also explored in cancer cell studies and display anticancer activities although the mechanism is unknown.1,

16

Among those metals, Pd(II) and Pt(II) are of

particular interest since their TSC complexes show comparable or higher


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anticancer activity to cisplatin, which is a widely prescribed antitumor drug.16-19 However the molecular targets of Pd(II) and Pt(II) compounds have not been clarified. In addition, it is important to note that TSCs have been shown to impact the activity of multiple enzymes and possible to chelate metals, which adds to the complexity of examining the mechanism in cells.9, 12, 20 TopoIIα is an established target for anticancer agents. It is one of six topoisomerases found in humans and belongs to the type IIA topoisomerase subfamily. Type II topoisomerases generate a transient enzyme-bound doublestranded DNA break and pass an intact DNA segment through the break before ligating the cleaved DNA. TopoIIα is a dimeric enzyme that requires Mg2+ to cleave and ligate DNA and ATP hydrolysis to perform DNA strand passage.20, 21 In contrast to the persistent expression of the other human isoform topoisomerase IIβ, TopoIIα is expressed predominantly in proliferating cells and involved in resolving topological problems during DNA replication and cell division.22 Compounds targeting TopoIIα are divided into two broad categories: catalytic inhibitors and interfacial poisons.21,

23-25

The catalytic inhibitors of

TopoIIα generally inhibit ATP hydrolysis in the N-terminus of the enzyme. Interfacial poisons target the TopoIIα catalytic domain, and convert transient double-stranded breaks to permanent breaks by blocking the ligation of cleaved DNA.23 Several research groups have examined the mechanism of how TSCs inhibit TopoIIα.

Molecular

modeling

suggested

that

N,N-dimethyl-2-(quinolin-2-

ylethylene) hydrazinecarbothioamide may be a catalytic inhibitor of TopoIIα by



binding to the ATP site with higher affinity than ATP itself.12,

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26

Studies on

quinoline-2-carboxaldehyde TSCs and their Cu(II) and Ni(II) complexes do not support a direct competition model because of the positive charge with the release of chloride after Cu(II)-TSC enters the cytoplasm.13 Our recent study showed that Cu(II) α-(N)-heterocyclic TSCs, Cu(II)-acetylpyridine-ethyl-TSC (CuYE) and Cu(II)-acetylpyrazine-methyl-TSC (CuZM) inhibit TopoIIα as noncompetitive catalytic inhibitors of ATP hydrolysis. We demonstrated both compounds inhibit ATP hydrolysis and increase DNA cleavage by TopoIIα.27 Our finding that Cu(II)-TSC acts as a noncompetitive TopoIIα catalytic inhibitor agrees with quinoline-2-carboxaldehyde TSCs studies, which suggest that Cu(II)-TSCs do not bind directly to the ATP binding site.13, 27 Cu(II)-TSC complexes demonstrate higher inhibitory activity than their corresponding TSC ligands.11,

13, 15

To find out what structural elements are

important for TSC inhibition of TopoIIα, we synthesized two series of TSCs without metal ions (referred to as ligands) and their Cu(II) and Pd(II) complexes. We also varied α-(N)-heterocyclic ring substrate segments, and side chain substitutions within the same series of α-(N)-heterocyclic TSCs. Those compounds were examined in TopoIIα-mediated plasmid DNA relaxation and cleavage assays respectively. Additionally, we performed ATPase, ligation, molecular modeling and ATP competition experiments. The following results explore the structure-activity relationship and clarify the mechanism of how TSC complexes stimulate TopoIIα-mediated DNA cleavage complexes.


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Experimental Procedures Enzyme and Materials. Human TopoIIα was expressed in yeast Saccharomyces cerevisiae and purified as described.27, 28 TopoIIα was stored in 50 mM Tris pH 7.8, 750 mM KCl, 40% glycerol and 0.5 mM dithiothreitol (DTT) as 1 mg/mL stock in liquid nitrogen. The recombinant plasmid pBR322 was amplified in Escherichia coli DH5α strain. The plasmid was purified following the protocol from Qiagen™ Plasmid Mega Kit and stored in ddH2O in liquid nitrogen as 1 mg/mL stock. Etoposide was purchased from Sigma and stored as 20 mM stock solutions in 100% dimethyl sulfoxide (DMSO) at 4C. Synthesis of Thiosemicarbazone Compounds. The benzoylpyridine-TSC (BZP) ligands and the Cu(II)-benzoylpyridine-TSC (CuBZP) complexes were synthesized by the method described previously.29 The synthesis of CuYE was described in previous studies.27 The synthesis of the acetylthiazole-TSC (ATZ) ligands was previously published.30 The synthesis and characterization of the other TSC compounds are listed in the Supporting Information. TSC compounds were dissolved in 100% DMSO and stored as 20 mM stock solutions at 4C. TopoIIα-mediated Plasmid Cleavage Assay. Plasmid DNA cleavage assays were performed similarly to methods describe previously.27 The reaction mixture contained 1 μg of human TopoIIα, 0.3 μg of pBR322 without or with 50 μM of compounds in a 20 μL reaction system with 10 mM Tris-HCl (pH 7.9), 5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA and 2.5% (v/v) glycerol. The reactions



were initiated by addition of TopoIIα, incubated at 37°C for 15 min and stopped by addition of 2 μL of 5% of SDS and 2 μL of 250 mM EDTA. Proteinase K was added to the reaction and incubated for 30 min at 45°C. The samples were then mixed with 2 μL of 6x agarose gel loading dye from Research Product International Corp. (Mount Prospect, IL) and subjected to electrophoresis in 1% TAE agarose gel with 0.5 μg/mL ethidium bromide at 110 Volts for 150 min. The results were then imaged with Bio-Rad Gel Doc XR+ or ChemiDoc MP imaging system. The data were quantified by Image Lab software from Bio-Rad (Hercules, California). TopoIIα-mediated Plasmid Relaxation Assay. Plasmid DNA relaxation assays were carried out as described previously.27 Briefly, reactions were prepared with 0.2 μg of human TopoIIα, 0.3 μg of pBR322 and 1 mM ATP in 10 mM Tris (pH 7.9), 175 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, and 2.5% Glycerol. TSC compounds with a concentration of 0.5, 1, 5, 10, or 50 μM were added. The reactions were incubated at 37°C for 30 min and terminated by addition of 3 μL of stop solution with 77.5 mM EDTA and 0.77% SDS. To digest the proteins, the reactions were then incubated with 0.08 mg/mL proteinase K at 45°C for 30 min. The samples were mixed with 2 μl of gel loading dye and run on a 1% agarose gel with TBE buffer. The gel was stained with 0.5 μg/mL ethidium bromide solution and visualized in Bio-Rad Gel Doc XR+ imaging system. Thin-Layer Chromatography-Based ATPase Assay. ATP hydrolysis was monitored using thin-layer chromatography (TLC) on a polyethylenimine (PEI) matrix (Merck KGaA, Darmstadt, Germany). Reactions were performed using


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conditions similar to relaxation reactions with minor adjustments. Reactions utilized DNA cleavage buffer with 1 mM ATP and were incubated at 37oC and 4 L samples were drawn at 30 min and spotted on the TLC plate. Reactions were run in the absence (2% DMSO as a control) or presence of Cu(II)- or Pd(II)-TSC complexes, as noted in the figure. The plate was then placed in 400 mM ammonium carbonate inside the TLC chamber and resolved. Separation of ADP from ATP was imaged using an AlphaImager system (Santa Clara, CA) and quantified using AlphaImager software. Data are displayed relative to the quantified amount of ADP formed by TopoIIα at time = 30 min in the absence of drug. For plotting and comparison purposes, this value was normalized to 1. Molecular Docking. The structure of the ATPase domain of human type IIA DNA topoisomerase used for docking was from the Protein Data Bank (PDB). It was a dimer form, and the PDB ID was 1zxm.31 The structure was prepared by adding hydrogen atoms with the condition pH=7.0, T=300K and a salt concentration of 0.1 M using the Protonate3D function of MOE 2018 (Chemical Computing Group Inc., Montreal, QC, Canada). The protein and the Cu(II)-TSC compounds (without Cl- anion) were parameterized using the AMBER10:EHT force field in MOE. In particular, the protein parameters were taken from Amber ff99SB,32 while the ligand bonded parameters were obtained with 2D Extended Hckel Theory,33 the VdW parameters were derived from GAFF34, and the charges from Bond Charge Increments. The receptor range in the protein was defined as all atoms of the following residues: R32, I33, Q35, R98, D152, D153, D154, and E155, as suggested by the previous study.13 Initial placement of the



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small ligand was done using the triangle matcher with London dG scoring, and refined by Rigid Receptor docking using GBVI/WSA scoring. Five docked poses of each Cu(II)-TSC were obtained, and the one with lower score was used in the analysis.

RESULTS The two series of α-(N)-heterocyclic TSCs ligands in this study have different α-(N)-heterocyclic

ring

substrate

segments:

benzoylpyridine

(BZP)

and

acetylthiazole (ATZ) as shown in Figure 1 and Table 1. Each ligand or metal complex also varied the side chain substitutions on the terminal nitrogen of the TSCs. Methyl, ethyl, tert-butyl, benzyl and phenyl TSCs were used to form the series of ligands with each different α-(N)-heterocyclic ring substrate, to give fifteen different ligands along with their Cu(II)-TSC and Pd(II)-TSC complexes (Supporting Information) (Table1). After synthesis and characterization, we examined their activities in TopoIIα-mediated plasmid DNA relaxation assay and cleavage assay respectively to explore the structure-activity relationship of the TSC complexes and their inhibition on TopoIIα. And we further investigated the mechanism of why Cu(II)-TSCs increase TopoIIα-mediated DNA cleavage by molecular docking and ATP competition assays. The Pd(II)-TSC complexes inhibit TopoIIα-mediated plasmid relaxation. The molecular targets of Pd(II)-TSC are unknown, although several Pd(II)-TSC complexes have been reported to exhibit anticancer activities.16, 19, 35 To examine


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whether Pd(II)-TSC complexes can inhibit TopoIIα in a purified enzyme system, the compounds were tested in TopoIIα-mediated plasmid DNA relaxation assay. As shown in lanes 13-17 of Figure 2A, Pd(II)-BZP-TSCs (PdBZP) with various side chain substitutions inhibited TopoIIα-mediated relaxation at 10 μM. In Figure 2A lanes 8-12, Cu(II)-BZP-TSCs (CuBZP) inhibited TopoIIα mediated DNA plasmid relaxation at 10 μM concentration while ligand BZP series showed no inhibition. We are the first to show that Pd(II)-TSC complexes inhibit TopoIIα. Also, our data are consistent with previous studies that Cu(II)-TSC complexes showed higher inhibition on TopoIIα than ligand TSCs.11, 13, 15, 28 To further test if other Pd(II)-TSC complexes inhibit TopoIIα, we synthesized novel TSCs with 5-membered thiazole ring structures such as ATZ (Supporting Information). As shown in Figure 2B, ATZ series compounds behave similarly to BZP complexes. Most of the Pd(II)-ATZ-TSCs (PdATZ) derivatives inhibited TopoIIα as presented in lanes 13-17 of Figures 2B with Pd(II)-ATZ-phenyl-TSCs (PdAP) exhibiting the weakest inhibition (lane 17 in Figure 2B). Ligand ATZs did not inhibit the activity of TopoIIα at 10 μM (lanes 3-7 in Figures 2B), but Cu(II)ATZ-TSCs (CuATZ) with each side chain substitution showed strong inhibition in lanes 8-12 of Figure 2B. In summary, at the concentration of 10 μM, the data clearly show that ligand TSCs do not inhibit TopoIIα, while all the Cu(II)-TSC complexes inhibit the enzyme. Further, the TopoIIα inhibition by the Pd(II)-TSC complexes varies with the phenyl substitution showing the weakest activity. CuAE and PdAE inhibit TopoIIα-mediated plasmid relaxation in a concentration-dependent manner. The ethyl derivatives of the ATZ series,



ATZ-ethyl-TSC, Cu(II)-ATZ-ethyl-TSC, Pd(II)-ATZ-ethyl-TSC (AE, CuAE and PdAE respectively) are measured in dose-dependent TopoIIα-mediated plasmid relaxation assays because the ethyl derivative of acetylpyridine-TSC (APY) was studied previously.27 As shown in Figure 3. The data clearly showed that both CuAE and PdAE started to inhibit TopoIIα at 5 μM. PdAE showed slightly higher inhibition at 5 μM than CuAE. Our result indicates that both CuAE and PdAE inhibit TopoIIα-mediated plasmid DNA relaxation in a dose-dependent manner just like CuYE.27 Both the Cu(II)-ethyl-TSC and Pd(II)-ethyl-TSC complexes inhibit ATP hydrolysis. TopoIIα requires ATP to perform strand passage and thus complete its catalytic cycle. CuYE is a TopoIIα catalytic inhibitor by inhibiting ATP hydrolysis.27 Here we studied a subset of the metal-TSC complexes to determine whether they can inhibit ATP hydrolysis. We examined each of the metal ethylTSC complexes (Cu and Pd) from BZP and ATZ groups and compared them to CuYE and DMSO. As shown in Figure 4, the level of ATP hydrolysis was measured by the presence of ADP. For comparison purposes, ADP levels in the presence of 1% DMSO (ND, open bar in Figure 4) at 30 min were set to 1, and compounds were assayed at 100 μM and compared to DMSO. The data in Figure 4 demonstrated in the presence of CuYE, CuBE(Cu(II)-BZP-ethyl-TSC), or CuAE (black bars in Figure 4), ATP hydrolysis dropped to below 30% of the control (DMSO). CuYE and CuBE inhibited ATP hydrolysis to less than 5%. In summary, our data suggest that the Cu(II)-TSC complexes studied here all display the ability to inhibit ATP hydrolysis.


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Since the Pd(II)-TSC complexes can also inhibit TopoIIα-mediated plasmid DNA relaxation, we examined their ability to inhibit ATP hydrolysis. In Figure 4, our data showed that Pd(II)-ethyl-TSC complexes (gray bars in Figure 4) inhibited

ATP

hydrolysis.

When

compared

with

their

Cu(II)-ethyl-TSC

counterparts, Pd(II)-ethyl-TSC complexes showed less inhibition of ATP hydrolysis with the exception of PdAE (Pd(II)-ATZ-ethyl-TSC). There is a statistically significant difference (p < 0.05) for APY and BZP but not ATZ when Cu and Pd are compared. Our data in Figures 3 and 4 strongly implicate the Cu(II) and Pd(II)-TSC complexes inhibit TopoIIα-mediated plasmid DNA relaxation through inhibiting ATP hydrolysis. Cu(II)-ethyl-TSC complexes reduce ATPase activity to a greater extent than Pd(II)-ethyl-TSC complexes in 2 of 3 cases with CuAE as an exception. PdAE showed higher inhibition on ATP hydrolysis in Figure 4. The data is consistent with dose-dependent relaxation assay in Figure 3, in which PdAE showed higher level of inhibition compared with CuAE. The Cu(II)-TSC complexes promote higher levels of TopoIIα-mediated plasmid cleavage when compared to ligand or Pd(II)-TSCs. Most catalytic inhibitors of TopoIIα do not increase TopoIIα-mediated DNA cleavage.23 However, CuYE and CuZM can not only inhibit ATP hydrolysis of TopoIIα, but also increase TopoIIα-mediated DNA cleavage.27 We wanted to investigate if the two series of TSCs in this study also increase the DNA cleavage mediated by TopoIIα and which elements in the TSC structure are important for stimulation. In the absence of ATP, TopoIIα cleaved supercoiled plasmid DNA to a linear form



as shown in lane 2 of Figures 5A, and 5C. Relative DNA cleavage was calculated by comparing the intensity of linear DNA in the presence of TSC compounds to linear DNA in the absence of drug (ND, linear bands in lane 2 of Figures 5A, and 5C). As shown in Figure 5A and Figure 5B, both CuBZPs (middle panel in Figure 5A and black bars in Figure 5B) and the PdBZP complexes (bottom panel in Figure 5A and gray bars in Figure 5B) increased the relative DNA cleavage, while the ligand TSCs alone had no impact on DNA cleavage. Further, CuBZPs induced higher levels of DNA cleavage when compared to PdBZPs. Additional studies on ATZs showed a similar trend. CuATZ (middle panel in Figure 5C and black bars in Figure 5D) exhibited strong stimulation of the DNA cleavage mediated by TopoIIα. PdATZ (bottom panel in Figure 5C and gray bars in Figure 5D) showed higher increase in DNA cleavage than corresponding ligand TSCs but lower increase than Cu(II) counterparts. As with BZPs, ligand ATZs (top panel in Figure 5C and open bars in Figure 5D) did not exhibit any increase in the relative DNA cleavage compared with the control (ND). The quantitative data in Figures 5B and 5D clearly showed that the Cu(II)TSC complexes induced 4- to 5-fold higher levels DNA cleavage when compared to the Pd(II)-TSC complexes. In some cases, Pd(II)-TSCs can increase the relative DNA cleavage 2- to 3-fold over control (ND), but several cases displayed baseline levels of DNA cleavage. CuAE and PdAE were compared to etoposide, a well-known interfacial poison for TopoIIα in dose-dependent DNA cleavage assays in Supplemental Figure S1. The level of DNA cleavage induced by CuAE elevated as the


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concentration increased. CuAE stimulated DNA cleavage complexes greater than etoposide. PdAE showed around 2-fold stimulation of DNA cleavage complexes overall. TSCs with different side chain substitutions on the terminal nitrogen exhibit slightly different activity against TopoIIα. Since the Cu(II)-TSC complexes are more effective in increasing TopoIIα-mediated DNA cleavage, we compared the influence of side chain substitutions of Cu(II)-TSCs. As shown in Figure 5B, CuBM, CuBE and CuBT (Refer to Table1 for acronyms) showed slightly higher stimulation of the DNA cleavage mediated by TopoIIα when compared with CuBB and CuBP. ATZs demonstrated similar trends as the BZP complexes. As shown in Figure 5D, CuAM, CuAE and CuAT had slightly higher activity when compared with CuAB and CuAP. CuBM, CuBE and CuBT all display statistically significantly higher levels of DNA cleavage when compared to the corresponding phenyl-TSC compound (p the substrate ring segments. Molecular modeling data



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(Figure 7) is consistent with results presented here since the substrate ring segments are pointing outside while the terminal nitrogen substitutions are pointing towards the protein. Also, the ATP competition assay supports a model where Cu(II)-TSCs binds near but outside the ATP binding pocket. At this point, it is unclear why Cu(II)-TSCs result in higher levels of DNA cleavage than Pd(II)TSCs. However, the data are consistent with previous studies that the Ni(II)-TSC complexes do not increase the DNA cleavage while Cu(II)-TSCs do.

13

One

possibility is that the redox property of Cu may contribute to the high activity of Cu(II)-TSC complexes. Additional studies with other metal ions, such as Fe, may give us insight on the role of metal ions in affecting TopoIIα-mediated DNA cleavage assay. Both the Cu(II) and Pd(II)-TSC complexes examined in this study behave similarly to the previous reported complex, CuYE,27 which suggests that they are noncompetitive inhibitors of ATP hydrolysis of TopoIIα and can stimulate TopoIIα-mediated DNA cleavage, though clearly to differing extents. Most catalytic inhibitors only inhibit the ATP hydrolysis, while interfacial poisons only increase the DNA cleavage complexes mediated by TopoIIα. Our metal-TSC complexes showed combined features of both catalytic inhibitors and interfacial poisons. Since interfacial poisons typically inhibit the ligation of double-stranded breaks, we examined the ligation of TopoIIα in the presence of metal-TSCs. Our data showed that metal-TSCs do not block DNA ligation (Supplement Figure S1). Results from molecular modeling data have consistently supported a model where Cu(II)-TSCs bind near the ATP pocket (Figure 8), which is consistent with


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the ligation results. Further, ATP competition assays (Figure 8) and previous ATPase data support a model where the TSCs are binding outside of the actual ATP binding pocket. Based upon this evidence, we hypothesize that binding of TSCs to TopoIIα mimics the binding of ATP to the enzyme and may cause conformational changes in the N-terminus, which is consistent with previous reports and our molecular modeling model.12,

13, 27

According to molecular modeling evidence,

Cu(II)-TSCs interact with key residues that may be involved in promoting dimerization of the N-terminal ATPase domain during DNA cleavage and strand passage.13 Previous reports indicate that compounds that affect the N-terminal ATPase domain can promote closing of the N-terminal “clamp” similar to ATP and cause increases in DNA cleavage.40,41,42 We suggest that this proposed drug:enzyme interaction near the ATP binding site may be what leads to the increase in DNA cleavage observed in the presence of Cu(II)-TSC complexes. Preliminary evidence indicates that Cu(II)-TSC complexes promote closure of the N-terminal clamp (manuscript in preparation, XJ/JED). Although the molecular docking model indicated what residues of TopoIIα binding to TSCs, the exact interaction surface of TSCs on TopoIIα remains unclear. Further biochemical and structural studies are needed to address the mechanism of how Cu(II)-TSC complexes interact with TopoIIα in the absence or presence of ATP and whether these complexes have similar effects on topoisomerase IIβ.

Supporting Information



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The Supporting Information is available free of charge on the ACS Publications website at DOI: …. Supplemental Methods: Plasmid DNA Ligation Assay Supplemental Methods: Synthesis of Thiosemicarbazone Compounds Figure S1: Concentration-dependent stimulation of TopoIIα-mediated plasmid DNA cleavage assay by CuAE, PdAE and etoposide. Figure S2: Cu(II)-etheyl-TSCs do not inhibit TopoIIα-mediated DNA ligation.

Funding This work was supported by funds from Faculty Development Grant from Tennessee Board of Regents (XJ and ECL), Faculty Research Grant of Tennessee Technological University (XJ), Tennessee Technological University URECA! Grants Program (LN, ARB, and MTS) and the Lipscomb University College of Pharmacy and Health Sciences. JTW was a participant in the Pharmaceutical Sciences Summer Research Program supported by the Lipscomb University College of Pharmacy and Health Sciences. ACKNOWLEDGEMENTS We thank Drs. Xuanzhi Zhan and Jeffery Rice for helpful discussions.

ABBREVIATIONS Thiosemicarbazone:

TSC;

benzoylpyridine-thiosemicarbazone:

acetylpyridine-thiosemicarbazone: BZP;

APY;

acetylthiazole-thiosemicarbazone:

ATZ; methyl-thiosemicarbazone: MTSC or M; ethyl-thiosemicarbazone: ETSC or


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E; tert-butyl-thiosemicarbazone: tBTSC or tB; benzyl-thiosemicarbazone, BzTSC or Bz; Phenyl-thiosemicarbazone: PTSC or P; Pd(II)-acetylpyridine-ethylthiosemicarbazone: PdYE; Cu(II)-bezoylpyridine-thiosemicarbazone: CuBZP; Pd(II)-bezoylpyridine-thiosemicarbazone: thiosemicarbazone:

CuATZ;

PdBZP;

Cu(II)-acetylthiazole-

Pd(II)-acetylthiazole-thiosemicarbazone:PdATZ;

Cu(II)-acetylpyridine-ethyl-thiosemicarbazone: [Cu(APY-ETSC)Cl]: CuYE; Cu(II)acetylpyrazine-methyl-thiosemicarbazone:

[Cu(APZ-MTSC)Cl]:

CuZM;

adenosine-5’-triphosphate: ATP; dithiothreitol: DTT; dimethyl sulfoxide: DMSO.



REFERENCES [1] Matesanz, A. I., and Souza, P. (2009) alpha-N-heterocyclic thiosemicarbazone derivatives as potential antitumor agents: a

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Figure Legends: Figure 1: The structures of α-(N)-heterocyclic TSCs with or without metal chelation. BZP and ATZ ligand only, Cu(II) and Pd(II) complex structures are shown. Additionally, Cu(II)-acetylpyridine-ethylthiosemicarbazone (CuYE) is shown on the right. Figure 2: Both Cu(II)-TSC and Pd(II)-TSC complexes inhibit plasmid DNA relaxation mediated by TopoIIα but not ligand TSC. BZP and ATZ series of TSCs were examined in TopoIIα-mediated plasmid DNA relaxation assay in Figures 2A, and 2B respectively. Lane 1, NT is the control without TopoIIα. Lane 2, ND is the DMSO control reaction in the absence of TSC compounds. TSC compounds with different side chain substitutions at 10 μM were tested as shown from lanes 3 to 17. Migration of relaxed DNA (R) and supercoiled DNA (SC) is denoted at right. The names of compounds were abbreviated. The first letter, B refers to BZP and A to ATZ. The second letter is as following M: methyl; E: ethyl; T: tertbutyl; B: benzyl; P: phenyl. Please check Table 1 for the full name of the compounds. Results are representative of three independent experiments. Figure 3: Concentration-dependent inhibition of TopoIIα-mediated plasmid DNA relaxation by CuAE and PdAE. Lanes 1 and 2 are controls without TopoIIα (NT) or without TSC compounds (ND, DMSO only). Increasing concentration of compound (0.5-10 μM) was incubated with TopoIIα as follows: lane 3-6, and CuAE lanes 7-10, PdAE. Migration of relaxed DNA (R) and supercoiled DNA (SC) is denoted at right. Results are representative of three independent experiments. Figure 4: Both Cu(II)-ethyl-TSC and Pd(II)-ethyl-TSC inhibit ATP hydrolysis of TopoIIα. CuBE, CuAE and their Pd(II) counterparts were examined in TLC based ATP hydrolysis assay. ND is the ADP level produced by ATP hydrolysis in the absence of TSC compounds (DMSO only) and was normalized to 1. Open bar: ND (DMSO only); Black bars: Cu(II)-ethyl-TSCs; Gray bars: Pd(II)-ethyl-TSCs. The error bars are the standard deviation of three independent experiments.


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Figure 5: Impact of TSC complexes on TopoII α-mediated DNA cleavage. The TopoIIα-mediated DNA cleavage reactions were incubated with 50 μM of BZPs (5A and 5B), ATZs (5C and 5D) or 1% DMSO (ND). NT (lane 1) is the control without TopoIIα. The intensity of linear DNA (L) in ND lanes was assigned to 1. The data in Figures 5B and 5D were quantified results of the intensity of linear DNA (L) compared to ND in Figures 5A and 5C, respectively. Open bars: Ligand TSC. Black bars: Cu(II)-TSC complexes. Gray bars: Pd(II)-TSC complexes. Lig, ligand-TSC; N, nicked DNA; L, linear DNA; SC, supercoiled DNA. Error bars represent the standard deviation of three or more independent experiments. Figure 6: Comparison of the effect of α-(N)-heterocyclic ring structures on TopoIIα-mediated DNA cleavage. The DNA cleavage mediated by TopoIIα was compared in the presence of different Cu(II)-ethyl-TSC complexes with different ring substrates APY, BZP, and ATZ. ND is DNA cleavage in the absence of any TSC compounds (DMSO only) and was normalized to 1. DNA cleavage in the presence of various TSC compounds was compared to ND. Error bars represent the standard deviation of three or more independent experiments. Figure 7: Snapshot of the docking structure of the ATPase domain of TopoIIα with (a) [Cu(YE)]+, (b) [Cu(AE)]+, and (c) [Cu(BE)]+. The Cu(II)-TSC complexes were in red, and the ANP was in blue. Figure 8: ATP cannot outcompete the Cu(II)-TSC complexes from binding to TopoIIα. DNA cleavage reactions with no TSC (ATP alone, black circles), 25 µM CuAE (red squares) or CuBE (blue triangles) were carried out in the presence of increasing concentrations of ATP (2 μM to 2 mM). DNA cleavage levels in the absence of ATP and TSCs were set to 1, and relative DNA cleavage levels were calculated for ATP and TSC reactions. ATP concentrations are plotted on a logarithmic scale. Error bars represent the standard deviation of three independent experiments. Table 1: Acronyms of α-(N)-heterocyclic thiosemicarbazones and their activities in TopoIIα-mediated DNA cleavage.



Figure 1: The structures of α-(N)-heterocyclic TSCs with or without metal chelation. BZP and ATZ ligand only, Cu(II) and Pd(II) complex structures are shown. Additionally, Cu(II)-acetylpyridineethylthiosemicarbazone (CuYE) is shown on the right. 194x182mm (300 x 300 DPI)


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Figure 2: Both Cu(II)-TSC and Pd(II)-TSC complexes inhibit plasmid DNA relaxation mediated by TopoIIα but not ligand TSC. BZP and ATZ series of TSCs were examined in TopoIIα-mediated plasmid DNA relaxation assay in Figures 2A, and 2B respectively. Lane 1, NT is the control without TopoIIα. Lane 2, ND is the DMSO control reaction in the absence of TSC compounds. TSC compounds with different side chain substitutions at 10 μM were tested as shown from lanes 3 to 17. Migration of relaxed DNA (R) and supercoiled DNA (SC) is denoted at right. The names of compounds were abbreviated. The names of compounds were abbreviated. The first letter B refers to BZP and A for ATZ. The second letter is as following, M: methyl; E: ethyl; T: tertbutyl; B: benzyl; P: phenyl. Please check Table 1 for the full name of the compounds. Results are representative of three independent experiments. 254x190mm (72 x 72 DPI)



Figure 3: Concentration-dependent inhibition of TopoIIα-mediated plasmid DNA relaxation by CuAE and PdAE. Lanes 1 and 2 are controls without TopoIIα (NT) or without TSC compounds (ND, DMSO only). Increasing concentration of compound (0.5-10 μM) was incubated with TopoIIα as follows: lane 3-6, and CuAE lanes 7-10, PdAE. Migration of relaxed DNA (R) and supercoiled DNA (SC) is denoted at right. Results are representative of three independent experiments. 254x190mm (72 x 72 DPI)


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Figure 4: Both Cu(II)-ethyl-TSC and Pd(II)-ethyl-TSC inhibit ATP hydrolysis of TopoIIα. CuBE, CuAE and their Pd(II) counterparts were examined in TLC based ATP hydrolysis assay. ND is the ADP level produced by ATP hydrolysis in the absence of TSC compounds (DMSO only) and was normalized to 1. Open bar: ND (DMSO only); Black bars: Cu(II)-ethyl-TSCs; Gray bars: Pd(II)-ethyl-TSCs. The error bars are the standard deviation of three independent experiments. 254x190mm (72 x 72 DPI)



254x190mm (72 x 72 DPI)


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Figure 5: Impact of TSC complexes on TopoII α-mediated DNA cleavage. The TopoIIα-mediated DNA cleavage reactions were incubated with 50 μM of BZPs (5A and 5B), ATZs (5C and 5D) or 1% DMSO (ND). NT (lane 1) is the control without TopoIIα. The intensity of linear DNA (L) in ND lanes was assigned to 1. The data in Figures 5B and 5D were quantified results of the intensity of linear DNA (L) compared to ND in Figures 5A and 5C, respectively. Open bars: Ligand TSC. Black bars: Cu(II)-TSC complexes. Gray bars: Pd(II)-TSC complexes. Lig, ligand-TSC; N, nicked DNA; L, linear DNA; SC, supercoiled DNA. Error bars represent the standard deviation of three or more independent experiments. 254x190mm (72 x 72 DPI)



Figure 6: Comparison of the effect of α-(N)-heterocyclic ring structures on TopoIIα-mediated DNA cleavage. The DNA cleavage mediated by TopoIIα was compared in the presence of different Cu(II)-ethyl-TSC complexes with different ring substrates APY, BZP, and ATZ. ND is DNA cleavage in the absence of any TSC compounds (DMSO only) and was normalized to 1. DNA cleavage in the presence of various TSC compounds was compared to ND. Error bars represent the standard deviation of three or more independent experiments. 61x86mm (300 x 300 DPI)


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Figure 7: Snapshot of the docking structure of the ATPase domain of TopoIIα with (a) [Cu(YE)]+, (b) [Cu(AE)]+, and (c) [Cu(BE)]+. The Cu(II)-TSC complexes were in red, and the ANP was in blue.



Figure 8: ATP cannot outcompete the Cu(II)-TSC complexes from binding to TopoIIα. DNA cleavage reactions with no TSC (ATP alone, black circles), 25 µM CuAE (red squares) or CuBE (blue triangles) were carried out in the presence of increasing concentrations of ATP (2 μM to 2 mM). DNA cleavage levels in the absence of ATP and TSCs were set to 1, and relative DNA cleavage levels were calculated for ATP and TSC reactions. ATP concentrations are plotted on a logarithmic scale. Error bars represent the standard deviation of three independent experiments. 172x120mm (150 x 150 DPI)


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Table 1 Benzoylpryridine Thiosemicarbazone (BZP) Benzoylpyridine Methylthiosemicarbazone Benzoylpyridine Ethylthiosemicarbazone Benzoylpyridine Tertbutylthiosemicarbazone Benzoylpyridine Benzylthiosemicarbazone Benzoylpyridine Phenylthiosemicarbazone Cu(II) Benzoylpyridine Methylthiosemicarbazone Cu(II) Benzoylpyridine Ethylthiosemicarbazone Cu(II) Benzoylpyridine Tertbutylthiosemicarbazone Cu(II) Benzoylpyridine Benzylthiosemicarbazone Cu(II) Benzoylpyridine Phenylthiosemicarbazone Pd(II) Benzoylpyridine Methylthiosemicarbazone Pd(II) Benzoylpyridine Ethylthiosemicarbazone Pd(II) Benzoylpyridine Tertbutylthiosemicarbazone Pd(II) Benzoylpyridine Benzylthiosemicarbazone Pd(II) Benzoylpyridine Phenylthiosemicarbazone

Acronym of BZP compounds with activity BM (-) BE (-) BT (-) BB (-) BP (-) CuBM (+++) CuBE (+++) CuBT (+++) CuBB (++) CuBP (++) PdBM (+) PdBE (+) PdBT (-) PdBB (-) PdBP (-)

Acetylthiazole Thiosemicarbazone (ATZ)

Acronym of ATZ compounds with activity

Acetylthiazole Methylthiosemicarbazone Acetylthiazole Ethylthiosemicarbazone Acetylthiazole Tertbutylthiosemicarbazone Acetylthiazole Benzylthiosemicarbazone Acetylthiazole Phenylthiosemicarbazone Cu(II) Acetylthiazole Methylthiosemicarbazone Cu(II) Acetylthiazole Ethylthiosemicarbazone Cu(II) Acetylthiazole Tertbutylthiosemicarbazone Cu(II) Acetylthiazole Benzylthiosemicarbazone Cu(II) Acetylthiazole Phenylthiosemicarbazone Pd(II) Acetylthiazole Methylthiosemicarbazone Pd(II) Acetylthiazole Ethylthiosemicarbazone Pd(II) Acetylthiazole Tertbutylthiosemicarbazone Pd(II) Acetylthiazole Benzylthiosemicarbazone Pd(II) Acetylthiazole Phenylthiosemicarbazone

AM (-)

*Relative activity in parentheses: Relative DNA cleavage increase > 7, +++ Relative DNA cleavage increase 4-7, ++ Relative DNA cleavage increase 2-4, + Relative DNA cleavage increase < 2, -


AE (-) AT (-) AB (-) AP (-) CuAM (+++) CuAE (+++) CuAT (++) CuAB (++) CuAP (++) PdAM (+) PdAE (+) PdAT (-) PdAB (-) PdAP (-)

Structural and Metal Ion Effects on Human Topoisomerase IIÎ±

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